Combining the advanced silicon etch system (ASETM) with electron beam lithography (EBL), we utilize the silicon sidewall scallops to fabricate 3D nano-fence matrixes for nanofluidic channels. We acquire the scallop size and the effective range of nano mask width for the formation of nano-fences under a fixed etch recipe. We then realize the controllability of fabricating silicon fences by simply varying the nano mask width. The fence matrixes make the micro-channel into integrated nano-channels, which will be very useful for fluid purification, segregation and bias application in certain fluid detection.